Immunoassays employing non-particulate chemiluminescent reagent

ABSTRACT

Methods and reagents are disclosed for conducting assays. Embodiments of the present methods and reagents are concerned with chemiluminescent reagents for determining the presence and/or amount of an analyte in a sample suspected of containing the analyte. The reagent is non-particulate and comprises a binding partner for the analyte and a chemiluminescent composition comprising an olefinic compound and a metal chelate. In embodiments of an assay, a combination is provided that comprises a sample suspected of containing the analyte, a chemiluminescent reagent as described above and a sensitizer reagent capable of generating singlet oxygen. The combination is subjected to conditions for binding of the analyte to the binding partner for the analyte. The sensitizer is activated and the amount of luminescence generated by the chemiluminescent composition is detected wherein the amount of luminescence is related to the amount of the analyte in the sample.

BACKGROUND

This invention relates to reagents, which are capable of generatingsignal, for use in methods, compositions and kits for determining ananalyte in a sample.

The clinical diagnostic field has seen a broad expansion in recentyears, both as to the variety of materials of interest that may bereadily and accurately determined, as well as the methods for thedetermination. Most methods involve generation of a signal in relationto the presence and/or amount of one or more analytes in a sample.Luminescent compounds, such as fluorescent compounds andchemiluminescent compounds, find wide application in the assay fieldbecause of their ability to emit light. Particles, such as latexparticles, liposomes and the like have been utilized in assays. Bothabsorptive dyes and dyes that impart fluorescent or chemiluminescentproperties have been incorporated into particles. In one particularapproach, particles that comprise one or more metal chelates such as,for example, lanthanide chelates, are employed for generating a signal.

An induced luminescence immunoassay is described in U.S. Pat. Nos.5,340,716 and 6,251,581, which disclosures are incorporated herein byreference. In one approach the assay uses a particle incorporating aphotosensitizer and a label particle incorporating a chemiluminescentcompound. The label particle is conjugated to a binding partner that iscapable of binding to an analyte to form a complex, or to another moietyto form a complex in relation to the presence of the analyte. If theanalyte is present, the photosensitizer and the chemiluminescentcompound come into close proximity. The photosensitizer generatessinglet oxygen and activates the chemiluminescent compound when the twolabels are in close proximity. The activated chemiluminescent compoundsubsequently produces light. The amount of light produced is related tothe amount of the complex formed, which in turn is related to the amountof analyte present. In one particular approach, the chemiluminescentparticles comprise one or more metal chelates such as, for example,lanthanide chelates.

In a variation of the induced luminescence method, a particulate supportis employed that comprises both (a) a photosensitizer capable uponirradiation of generating singlet oxygen and (b) a chemiluminescentcompound capable of being activated by singlet oxygen. The methods allowfor generating delayed luminescence, which can be realized uponirradiation of the support. The methods have application to thedetermination of an analyte in a medium suspected of containing theanalyte. One method comprises subjecting a medium suspected ofcontaining an analyte to conditions under which a complex of bindingpartners is formed in relation to the presence of the analyte anddetermining whether the complex has formed by employing as a label aparticulate composition having both chemiluminescent and photosensitizerproperties. Upon activation of the photosensitizer property, singletoxygen is generated and activates the chemiluminescent property. Suchcompositions and methods are described in U.S. Pat. No. 5,709,994, therelevant disclosure of which is incorporated herein by reference.

SUMMARY

One embodiment of the present invention is a chemiluminescent reagentfor determining the presence and/or amount of an analyte in a samplesuspected of containing the analyte. The chemiluminescent reagent isnon-particulate and comprises a binding partner for the analyte and achemiluminescent composition comprising an olefinic compound and a metalchelate.

Another embodiment of the present invention is a method for determiningthe presence and/or amount of an analyte in a sample suspected ofcontaining the analyte. A combination is provided that comprises thesample, a chemiluminescent reagent as described above and a sensitizerreagent capable of generating singlet oxygen. The combination issubjected to conditions for binding of the analyte to the bindingpartner for the analyte. The sensitizer is activated and the amount ofluminescence generated by the chemiluminescent composition is detectedwherein the amount of luminescence is related to the amount of theanalyte in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of a scheme for producing a chemiluminescentreagent in accordance with present embodiments.

FIG. 2 is a graph depicting a calibration curve for an assay for TSHutilizing a chemiluminescent reagent in accordance with the presentembodiments.

FIG. 3 is a tabular depiction of the results of an assay for TSHutilizing a chemiluminescent reagent in accordance with the presentembodiments and showing the separation of signal obtained betweencalibrators.

FIG. 4 is a graph depicting the results of a stability study of achemiluminescent reagent in accordance with the present embodiments.

FIG. 5 is a graph depicting the results of a stability study of achemiluminescent reagent in accordance with the present embodiments.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS General Discussion

Embodiments of the present methods and reagents are concerned withchemiluminescent reagents for determining the presence and/or amount ofan analyte in a sample suspected of containing the analyte. The reagentis non-particulate and comprises a binding partner for the analyte and achemiluminescent composition comprising an olefinic compound and a metalchelate. The chemiluminescent reagent in accordance with the presentembodiments exhibits good solubility in an aqueous medium such as anaqueous assay medium. The non-particulate chemiluminescent reagent hasenhanced stability for use in assays for detection of analytes;furthermore, the reagent exhibits good signal response to changes in theconcentration of analyte. When used in an assay, the non-particulatechemiluminescent reagent in accordance with the present embodiments issoluble in aqueous assay media and provides maximized performanceincluding accuracy and sensitivity as well as stability of signalproduced.

Performance of a particular assay format at the low end of the medicaldecision range can be monitored by monitoring the difference in theamount of signal obtained for calibrators spanning the suspectedconcentration range of interest of the analyte. A large difference orseparation between the signal for calibrators such as, for example,calibrator L1 and calibrator L2 or calibrator L2 and calibrator L3, isdesired. For example, six calibrators may be employed, arbitrarily namedL1-L6. Signal to noise ratio may be evaluated by determining an amountof signal using a calibrator that contains no analyte, arbitrarilydesignated calibrator L1 (background), and the amount of signal obtainedfor a calibrator containing a first known amount of analyte above zero,arbitrarily designated calibrator L2. This evaluation may also includedetermining an amount of signal using calibrator L1 and the amount ofsignal for a calibrator containing a second known amount of analyteabove zero, arbitrarily designated L3. Such an evaluation may alsoinclude such determination using calibrators L4, L5, L6 and so forth.The embodiments discussed herein provide for better performance in anassay for an analyte compared to reagents not in accordance with thepresent embodiments.

A large difference between the signal for calibrators, e.g., calibratorL1 and calibrator L2, or calibrator L1 and calibrator L6, is desired.For good sensitivity in the medical decision range, the difference inthe signal detected between calibrator L1 and calibrator L2 is at leastabout 50%, at least about 75%, at least about 90%, at least about 100%,at least about 125%, at least about 150%, at least about 175%, at leastabout 200%, at least about 225%, at least about 250%, at least about275%, at least about 300%, at least about 325%, at least about 350%, atleast about 375%, at least about 400%, at least about 425%, and soforth. In some embodiments the signal detected for calibrator L6 is atleast about 10 times, at least about 20 times, at least about 30 times,at least about 40 times, at least about 50 times, at least about 60times, at least about 70 times, at least about 80 times, at least about90 times, at least about 100 times, greater than the signal detected forcalibrator L1. Depending on the assay format, the difference in signalmay be an increase in signal or a decrease in signal. Typically, theresults of the assays using the calibrators are presented in a graphformat wherein the amount of signal is plotted against the concentrationof the calibrators. In accordance with embodiments of the presentinvention the slope of the line between calibrator L1 and calibrator L2generally is steeper compared with results obtained with assay reagentsnot in accordance with the present embodiments. Furthermore, the slopeof the line from calibrator L1 to calibrator L6 is usually steepercompared with results obtained with assay reagents not in accordancewith the present embodiments.

As mentioned above, embodiments of the present chemiluminescent reagentsare non-particulate. As such, the reagents are distinguished from thoseemployed in the aforementioned known induced luminescence assays, whichemploy particulate chemiluminescent reagents. The presentchemiluminescent reagents exhibit good solubility in aqueous media. Inembodiments of the chemiluminescent reagents, a binding partner for theanalyte is covalently or non-covalently bound to a chemiluminescentcomposition comprising an olefinic compound and a metal chelate.

The binding partner for the analyte may be covalently bound to thechemiluminescent composition by a bond. On the other hand, the bindingpartner for the analyte may be covalently bound to the chemiluminescentcomposition by a linking group. In some embodiments, the linking groupis hydrophilic. The term “hydrophilic” or “hydrophilicity” refers to amoiety that is polar and thus prefers polar molecules and prefers polarsolvents. Hydrophilic molecules have an affinity for other hydrophilicmoieties compared to hydrophobic moieties. The degree of hydrophilicityis controlled by the number of heteroatoms in the linking group.

In some embodiments the linking group is a macromolecule and may bepolymeric. The polymeric macromolecule is generally about 1 to about10,000 monomer units or more in length, or about 10 to about 10,000monomer units in length, or about 100 to about 10,000 monomer units inlength, or about 500 to about 10,000 monomer units in length, or about1,000 to about 10,000 monomer units in length, or about 2,000 to about10,000 monomer units in length, or about 3,000 to about 10,000 monomerunits in length, or about 5,000 to about 10,000 monomer units in length,or about 10 to about 8,000 monomer units in length, or about 100 toabout 8,000 monomer units in length, or about 1,000 to about 8,000monomer units in length, or about 100 to about 7,000 monomer units inlength and the like. The number of monomer units depends on the numberof atoms in the monomer unit chain, the composition of the monomer unit,and so forth.

The molecular weight of the polymeric macromolecule is at least about2,000. The molecular weight may be about 2,000 to about 10,000,000 ormore, or about 2,000 to about 8,000,000, or about 2,000 to about6,000,000, or about 2,000 to about 5,000,000 or about 2,000 to about4,000,000, or about 2,000 to about 3,000,000 or about 2,000 to about2,000,000, or about 2,000 to about 1,000,000, or about 5,000 to about10,000,000 or more, or about 5,000 to about 8,000,000, or about 5,000 toabout 6,000,000, or about 5,000 to about 5,000,000 or about 5,000 toabout 4,000,000, or about 5,000 to about 3,000,000 or about 5,000 toabout 2,000,000, or about 5,000 to about 1,000,000, or about 10,000 toabout 10,000,000 or more, or about 10,000 to about 8,000,000, or about10,000 to about 6,000,000, or about 10,000 to about 5,000,000 or about10,000 to about 4,000,000, or about 10,000 to about 3,000,000 or about10,000 to about 2,000,000, or about 10,000 to about 1,000,000, and thelike.

The polymeric macromolecule may be linear or branched or a combinationthereof. A linear polymer comprises a linear chain of atoms and abranched polymer comprises a branched chain of atoms. Each atom of thelinear chain may have one or more substituents in place of hydrogen. Insome embodiments the polymer may be a copolymer comprising more than onetype of monomer unit. The relationship of the different monomer units inthe polymer may be alternating, random, periodic and the like and mayalso be in a block copolymer arrangement where blocks of repeatingmonomer units form the polymer chain.

In some embodiments one or more of the monomer units of the polymericmacromolecule comprise carbon atoms and one or more heteroatoms such as,for example, oxygen, sulfur, nitrogen, phosphorus, and the like. Themonomer units may comprise about 2 to about 50 atoms or more, or 5 toabout 50 atoms, or about 10 to about 50 atoms, or about 20 to about 50atoms, or about 30 to about 50 atoms, or about 2 to about 40 atoms ormore, or 5 to about 40 atoms, or about 10 to about 40 atoms, or about 20to about 40 atoms, or about 30 to about 40 atoms, or about 2 to about 30atoms or more, or 5 to about 30 atoms, or about 10 to about 30 atoms, orabout 20 to about 30 atoms, not counting hydrogen and may comprise achain of from 2 to about 30 atoms, or about 5 to about 30 atoms or more,or 10 to about 30 atoms, or about 15 to about 30 atoms, or about 20 toabout 30 atoms, or about 2 to about 25 atoms or more, or 5 to about 25atoms, or about 10 to about 25 atoms, or about 15 to about 25 atoms, orabout 20 to about 25 atoms, or about 2 to about 20 atoms or more, or 5to about 20 atoms, or about 10 to about 20 atoms, or about 15 to about20 atoms, or about 2 to about 15 atoms, or about 5 to about 15 atoms, orabout 10 to about 15 atoms, each independently selected from the groupnormally consisting of carbon, oxygen, sulfur, nitrogen, andphosphorous.

The number of heteroatoms in a monomer unit of the polymericmacromolecule may range from about 0 to about 20, or about 0 to about15, or about 0 to about 10, or about 0 to about 5, or about 1 to about20, or about 1 to about 15, or about 1 to about 10, or about 1 to about5, or about 2 to about 20, or about 2 to about 15, or about 2 to about10, or about 2 to about 5, or about 5 to about 20, or about 5 to about15, or about 5 to about 10, and the like. In some embodiments, thenumber of heteroatoms is sufficient to render the linking grouphydrophilic and enhance the solubility of the chemiluminescent reagentin accordance with the present embodiments. In this regard, the numberof heteroatoms in the monomer unit of the polymeric macromolecule mayrange from about 1 to about 20, or about 1 to about 15, or about 1 toabout 10, or about 1 to about 5, or about 2 to about 20, or about 2 toabout 15, or about 2 to about 10, or about 2 to about 5, or about 5 toabout 20, or about 5 to about 15, or about 5 to about 10, and the like.When heteroatoms are present, oxygen is normally present as oxo or oxy,bonded to carbon, sulfur, nitrogen or phosphorous, nitrogen is normallypresent as azo, cyano, isocyano, nitro, nitroso, amido or amino,normally bonded to carbon, oxygen, sulfur or phosphorous; sulfur isanalogous to oxygen; while phosphorous is bonded to carbon, sulfur,oxygen or nitrogen, usually as phosphonate and phosphate mono- ordiester.

Common functionalities in forming a covalent bond between the linkinggroup and the molecule to be conjugated are alkylamine, amidine,thioamide, ether, urea, thiourea, guanidine, azo, thioether andcarboxylate, sulfonate, and phosphate esters, amides and thioesters. Forthe most part, when a linking group has a linking functionality(functionality for reaction with a moiety) such as, for example, anon-oxocarbonyl group including nitrogen and sulfur analogs, a phosphategroup, an amino group, alkylating agent such as halo or tosylalkyl, oxy(hydroxyl or the sulfur analog, mercapto) oxocarbonyl (e.g., aldehyde orketone), or active olefin such as a vinyl sulfone or α-, β-unsaturatedester, these functionalities are linked to amine groups, carboxylgroups, active olefins, alkylating agents, e.g., bromoacetyl. Where anamine and carboxylic acid, or its nitrogen derivative or phosphoric acidderivative, are linked, amides, amidines and phosphoramides are formedrespectively. Where mercaptan and activated olefin are linked,thioethers are formed. Where a mercaptan and an alkylating agent arelinked, thioethers are formed. Where aldehyde and an amine are linkedunder reducing conditions, an alkylamine is formed. Where a ketone oraldehyde and a hydroxylamine (including derivatives thereof where asubstituent is in place of the hydrogen of the hydroxyl group) arelinked, an oxime functionality (═N—O—) is formed. Where a carboxylicacid or phosphate acid and an alcohol are linked, esters are formed.

In some embodiments, the linking group is not a macromolecule and has amolecular weight less than about 2000, or less than about 1500, or lessthan about 1000, or less than about 500, or the like. Such linkinggroups may comprise about 2 to about 200 atoms, or 4 to about 150 atoms,or about 5 to about 100 atoms, not counting hydrogen and may comprise achain of from 2 to about 100 atoms, or 3 to about 90 atoms, or about 4to about 80 atoms, or about 5 to about 70 atoms, or the like, eachindependently selected from the group normally consisting of carbon,oxygen, sulfur, nitrogen, and phosphorous. The number of heteroatoms insuch linking groups is dependent on the size of the linking group andwill normally range from about 0 to about 50, 1 to about 45, or about 2to about 40. The heteroatoms may be in the forms indicated above in thediscussion concerning macromolecular linking groups. In someembodiments, the number of heteroatoms is sufficient to render thelinking group hydrophilic and enhance the solubility of the resultantcomposition in accordance with the present embodiments. In this regard,the number of heteroatoms in the linking group depends on the size ofthe linking group and may range from about 5 to about 50, or about 10 toabout 50, or about 15 to about 50, or about 5 to about 40, or about 10to about 40, or about 15 to about 40, or about 5 to about 30, or about10 to about 30, or about 15 to about 30, or about 5 to about 25, orabout 10 to about 25, and the like.

A polymeric macromolecule as a linking group may be anaturally-occurring material or a synthetic construct. In someembodiments the polymeric macromolecular linking group is a polypeptide.Examples of polypeptides, by way of illustration and not limitation,include proteins such as, e.g., albumins, gammaglobulins,immunoglobulins, hemocyanins, synthetic polypeptides, and the like.Examples of other polymeric macromolecules, by way of illustration andnot limitation, include dendrimers, polymeric carboxylates (e.g.,polyaspartic acid, polyglutamic acid, polygalacturonic acid,polymethacrylic acid, etc.), polymeric amines (e.g., polyethylene amine,polylysine, polyglutamine, polyethylene imine, polyallylamine, etc.),polymeric ethers (polyethyleneglycols or polyethylene oxide, etc.),polymeric thioethers (e.g., polyethylene thioethers, etc.), polymericsulfhydryls (e.g. polycysteine) and so forth.

The binding partner for the analyte may be bound to a chemiluminescentcomposition in a number of different ways, some of which are discussedbelow by way of illustration and not limitation. In some embodiments thelinking group is a protein and the components of the chemiluminescentcomposition, namely, an olefinic compound and a metal chelate, are eachbound to the protein, to which a binding partner for the analyte is alsobound. This embodiment is illustrated as follows:

wherein Ab is a binding partner for the analyte (in this example, anantibody), L is protein linking group, MC is metal chelate and OC isolefinic compound and a, b and c are independently an integer of 1 toabout 10, or 1 to about 9, or 1 to about 8, or 1 to about 7, or 1 toabout 6, or 1 to about 5, or 1 to about 4, or 1 to about 3, or 1 toabout 2, or 2 to about 10, or 2 to about 9, or 2 to about 8, or 2 toabout 7, or 2 to about 6, or 2 to about 5, or 2 to about 4, or 2 toabout 3, or about 3 to about 10, or about 3 to about 9, or about 3 toabout 8, or about 3 to about 7, or about 3 to about 6, or about 3 toabout 5, or about 3 to about 4, for example. Various functionalitiessuch as amine, carboxyl and the like are present on the protein forlinking to MC, OC and Ab and, as can be seen, multiple molecules of MC,OC and Ab may be bound to the protein. The number of molecules of eachthat may be bound is dependent on the size of the protein, the size ofMC and OC and the size of the antibody.

In some embodiments the linking group is a protein and the components ofthe chemiluminescent composition, namely, an olefinic compound and ametal chelate, are each bound to the same molecule of the protein. Theprotein has one or more molecules of a member of a specific binding pairbound thereto. The binding partner for the analyte has one or moremolecules of the other member of the specific binding pair boundthereto. The binding of the members of the specific binding pair resultsin the non-covalent binding of the binding partner for the analyte tothe protein. This embodiment is illustrated as follows:

wherein Ab is a binding partner for the analyte (in this example, anantibody), L is protein linking group, MC is metal chelate, OC isolefinic compound, SBPM1 and SBPM2 are complementary members of aspecific binding pair, ═ is a non-covalent bond, and a, b, c and d areindependently an integer of 1 to about 10, or 1 to about 9, or 1 toabout 8, or 1 to about 7, or 1 to about 6, or 1 to about 5, or 1 toabout 4, or 1 to about 3, or 1 to about 2, or 2 to about 10, or 2 toabout 9, or 2 to about 8, or 2 to about 7, or 2 to about 6, or 2 toabout 5, or 2 to about 4, or 2 to about 3, or about 3 to about 10, orabout 3 to about 9, or about 3 to about 8, or about 3 to about 7, orabout 3 to about 6, or about 3 to about 5, or about 3 to about 4, forexample. Various functionalities such as amine, carboxyl and the likeare present on the protein for linking to MC, OC and SBPM1 and, as canbe seen, multiple molecules of MC, OC and SBPM1 may be bound to theprotein. The number of molecules of each that may be bound is dependenton the size of the protein, the size of MC, OC, SBPM1 and SBPM2 and thesize of the antibody.

In some embodiments the linking group is a protein and the components ofthe chemiluminescent composition, namely, an olefinic compound and ametal chelate, are each bound to the same molecule of the protein. Theprotein has one or more molecules of a member of a specific binding pairbound thereto as well as one or more molecules of the antibody. OC andMC each independently have one or more molecules of the other member ofthe specific binding pair bound thereto. The binding of the members ofthe specific binding pair results in the non-covalent binding of OC andMC to the protein. This embodiment is illustrated as follows:

wherein Ab is a binding partner for the analyte (in this example, anantibody), L is protein linking group, MC is metal chelate, OC isolefinic compound, SBPM1 and SBPM2 are complementary members of aspecific binding pair, ═ is a non-covalent bond, and a, b, c and d areindependently an integer of 1 to about 10, or 1 to about 9, or 1 toabout 8, or 1 to about 7, or 1 to about 6, or 1 to about 5, or 1 toabout 4, or 1 to about 3, or 1 to about 2, or 2 to about 10, or 2 toabout 9, or 2 to about 8, or 2 to about 7, or 2 to about 6, or 2 toabout 5, or 2 to about 4, or 2 to about 3, or about 3 to about 10, orabout 3 to about 9, or about 3 to about 8, or about 3 to about 7, orabout 3 to about 6, or about 3 to about 5, or about 3 to about 4, forexample. Various functionalities such as amine, carboxyl and the likeare present on the protein for linking to MC, OC and SBPM1 and, as canbe seen, multiple molecules of MC, OC and SBPM1 may be bound to theprotein. The number of molecules of each that may be bound is dependenton the size of the protein, the size of MC, OC, SBPM1 and SBPM2 and thesize of the antibody. It should be noted that in the above embodimentthe specific binding pair to which SBPM1 and SBPM2 belong may be thesame for attachment of OC and MC, or two different specific bindingpairs may be employed, one for OC and one for MC.

In some embodiments the linking group is a protein and the components ofthe chemiluminescent composition, namely, an olefinic compound and ametal chelate, are each bound to the same molecule of the protein. Theprotein has one or more molecules of a member of a specific binding pairbound thereto. OC and MC each independently have one or more moleculesof the other member of the specific binding pair bound thereto. Thebinding of the members of the specific binding pair results in thenon-covalent binding of OC and MC to the protein. The binding partnerfor the analyte also has one or more molecules of the other member ofthe specific binding pair bound thereto. The binding of the members ofthe specific binding pair results in the non-covalent binding of thebinding partner for the analyte to the protein. This embodiment isillustrated as follows:

wherein Ab is a binding partner for the analyte (in this example, anantibody), L is protein linking group, MC is metal chelate, OC isolefinic compound, SBPM1 and SBPM2 are complementary members of aspecific binding pair, ═ is a non-covalent bond, and a, b, c and d areindependently an integer of 1 to about 10, or 1 to about 9, or 1 toabout 8, or 1 to about 7, or 1 to about 6, or 1 to about 5, or 1 toabout 4, or 1 to about 3, or 1 to about 2, or 2 to about 10, or 2 toabout 9, or 2 to about 8, or 2 to about 7, or 2 to about 6, or 2 toabout 5, or 2 to about 4, or 2 to about 3, or about 3 to about 10, orabout 3 to about 9, or about 3 to about 8, or about 3 to about 7, orabout 3 to about 6, or about 3 to about 5, or about 3 to about 4, forexample. Various functionalities such as amine, carboxyl and the likeare present on the protein for linking to SBPM1 and, as can be seen,multiple molecules of SBPM1 may be bound to the protein. The number ofmolecules of each that may be bound is dependent on the size of theprotein, the size of MC, OC, SBPM1 and SBPM2 and the size of theantibody. It should be noted that in the above embodiment the specificbinding pair to which SBPM1 and SBPM2 belong may be the same forattachment of OC, MC and the antibody or two or three different specificbinding pairs may be employed, one for OC, one for MC and one for theantibody.

In some embodiments the linking group is a protein and the components ofthe chemiluminescent composition, namely, an olefinic compound and ametal chelate, are respectively bound to different molecules of theprotein. The protein has one or more molecules of a member of a specificbinding pair bound thereto. The binding partner for the analyte has oneor more molecules of the other member of the specific binding pair boundthereto. The binding of the members of the specific binding pair resultsin the non-covalent binding of the binding partner for the analyte tothe protein. This embodiment is illustrated as follows:

wherein Ab is a binding partner for the analyte (in this example, anantibody), L is protein linking group, MC is metal chelate, OC isolefinic compound, SBPM1 and SBPM2 are complementary members of aspecific binding pair, ═ is a non-covalent bond, and a, b, c and d areindependently an integer of 1 to about 10, or 1 to about 9, or 1 toabout 8, or 1 to about 7, or 1 to about 6, or 1 to about 5, or 1 toabout 4, or 1 to about 3, or 1 to about 2, or 2 to about 10, or 2 toabout 9, or 2 to about 8, or 2 to about 7, or 2 to about 6, or 2 toabout 5, or 2 to about 4, or 2 to about 3, or about 3 to about 10, orabout 3 to about 9, or about 3 to about 8, or about 3 to about 7, orabout 3 to about 6, or about 3 to about 5, or about 3 to about 4, forexample. Various functionalities such as amine, carboxyl and the likeare present on the protein for linking to MC, OC and SBPM1 and, as canbe seen, multiple molecules of MC, OC and SBPM1 may be bound to theprotein. The number of molecules of each that may be bound is dependenton the size of the protein, the size of MC, OC, SBPM1 and SBPM2 and thesize of the antibody. It should be noted that in the above embodimentthe specific binding pair to which SBPM1 and SBPM2 belong may be thesame for attachment of OC and MC or two different specific binding pairsmay be employed, one for OC and one for MC. It should also be noted thatin the above embodiment L may be the same or different for OC and MC.

In some embodiments the linking group is a protein and the components ofthe chemiluminescent composition, namely, an olefinic compound and ametal chelate, are respectively bound to different molecules of theprotein by means of non-covalent binding. The protein has one or moremolecules of a member of a specific binding pair bound thereto. Thebinding partner for the analyte has one or more molecules of the othermember of the specific binding pair bound thereto. The binding of themembers of the specific binding pair results in the non-covalent bindingof the binding partner for the analyte to the protein. OC and MC alsoeach independently have one or more molecules of the other member of thespecific binding pair bound thereto. The binding of the members of thespecific binding pair results in the non-covalent binding of OC and MCto the protein. This embodiment is illustrated as follows:

wherein Ab is a binding partner for the analyte (in this example, anantibody), L is protein linking group, MC is metal chelate, OC isolefinic compound, SBPM1 and SBPM2 are complementary members of aspecific binding pair, ═ is a non-covalent bond, and a, b, c and d areindependently an integer of 1 to about 10, or 1 to about 9, or 1 toabout 8, or 1 to about 7, or 1 to about 6, or 1 to about 5, or 1 toabout 4, or 1 to about 3, or 1 to about 2, or 2 to about 10, or 2 toabout 9, or 2 to about 8, or 2 to about 7, or 2 to about 6, or 2 toabout 5, or 2 to about 4, or 2 to about 3, or about 3 to about 10, orabout 3 to about 9, or about 3 to about 8, or about 3 to about 7, orabout 3 to about 6, or about 3 to about 5, or about 3 to about 4, forexample. Various functionalities such as amine, carboxyl and the likeare present on the protein for linking to MC, OC and SBPM1 and, as canbe seen, multiple molecules of MC, OC and SBPM1 may be bound to theprotein. The number of molecules of each that may be bound is dependenton the size of the protein, the size of MC, OC, SBPM1 and SBPM2 and thesize of the antibody. It should be noted that in the above embodimentthe specific binding pair to which SBPM1 and SBPM2 belong may be thesame for attachment of OC, MC and antibody or two or three differentspecific binding pairs may be employed, one for OC and one for MC andone for the antibody. It should also be noted that in the aboveembodiment L may be the same or different for OC and MC.

The olefinic compound is one that is capable of reaction with singletoxygen. In some embodiments, reaction of olefins with singlet oxygen is2+2 addition to form a dioxetane. Suitable olefinic compounds usuallyhave no saturated C—H group attached to an olefinic carbon exceptunreactive bridgehead carbons and, in some embodiments, have one or moreelectron donating groups directly attached to the olefinic carbon or inconjugation with the olefin. Dioxetanes can dissociate spontaneously orby heating with spontaneous chemiluminescence, or the carbonyl groupsthat are formed can be formed as part of a fluorescent group or becapable of undergoing subsequent reactions that lead to a fluorescentmolecule. Alternatively, this dissociation reaction can lead toseparation of a quenching group from a fundamentally fluorescent groupthat thereby regains its fluorescent property.

In some embodiments, reaction of singlet oxygen with olefins is 4+2cycloaddition with dienes, usually aromatic compounds such asnaphthalenes, anthracenes, oxazoles, furans, indoles, and the like. Sucha reaction leads initially to an endoperoxide. In some casesendoperoxides can rearrange to active esters or anhydrides that arecapable of reaction with a suitably placed group to provide a lactone orlactam that can be fluorescent. Alternatively, the endoperoxides mayoxidize a fluorescent or chemiluminescent compound precursor.Endoperoxides can also dissociate spontaneously or on heating withchemiluminescent emission or oxidize a fluorescent leuco dye.

In some embodiments, reaction of singlet oxygen with olefins is the“ene” reaction that produces an allylhydroperoxide. Suitable olefinshave a reactive saturated C—H attached to an olefinic carbon. Thisproduct can react with an active ester in the same molecule to form adioxetanone that can spontaneously or by heating dissociate withchemiluminescent emission.

In general, olefins of interest are those that undergo a chemicalreaction upon reaction with singlet oxygen to form a metastable reactionproduct, usually a dioxetane or endoperoxide, which is capable ofdecomposition with the simultaneous or subsequent emission of light,usually within the wavelength range of 250 to 1200 nm. Preferred areelectron rich olefins usually containing electron-donating groups.Exemplary of such electron rich olefins are enol ethers, enamines,9-alkylidene-N-alkylacridans, arylvinylethers, 1,4-dioxenes,1,4-thioxenes, 1,4-oxazines, arylimidazoles, 9-alkylidene-xanthanes andlucigenin.

Examples of suitable electron rich chemiluminescent olefins are setforth in U.S. Pat. No. 5,709,994, the relevant disclosure of which isincorporated herein by reference. Such olefins generally have anelectron-donating group in conjugation with the olefin.

The dioxetanes may be luminescent alone or in conjunction with afluorescent energy acceptor. Enol ethers are examples of such olefins.In some embodiments, the enol ether compounds will have at least onearyl group bound to the olefinic carbons where the aryl ring issubstituted with an electron donating group at a position that increasesthe reactivity of the olefin to singlet oxygen and/or impartsfluorescence to the product of dissociation of the resultant dioxetane.The electron-donating group can be, for example, hydroxyl, alkoxy,disubstituted amino, alkyl thio, furyl, pyryl, etc. Preferably, the enolethers have an electron-donating group bound directly to an olefiniccarbon.

Enamines are another example of such olefins. In general, usefulenamines will be governed by the rules set forth above for enol ethers.Another family of chemiluminescers is the 2,4,5-triphenylimidazoles,with lophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino and para-methoxy substituents. Otherchemiluminescent olefins that satisfy the requirements given above maybe found in European Patent Application No. 0,345,776.

In addition to the olefinic compound, the chemiluminescent compositioncomprises a complex of a metal and one or more chelating agents.Examples of metals that form part of the complex include, for example,rare earth metals, metals in Group VIII, and the like. The rare earthmetals comprise the lanthanoids (lanthanide metals) (the 15 elementsfrom lanthanum to lutetium, atomic numbers 57-71). The rare earth metalsof particular interest include europium, terbium, dysprosium andsamarium. The metals of Group VIII a particular interest include osmiumand ruthenium. In some embodiments, rare earth metals have an oxidationstate of plus three, ruthenium has an oxidation state of plus two andosmium has an oxidation state of plus two. In certain embodiments themetal is selected from the group consisting of europium, terbium,dysprosium, samarium, osmium and ruthenium. In some embodiments themetal is at least hexacoordinated; however, the metal may beoctacoordinated or more highly coordinated depending on the metalchelating agent.

The metal chelating agent is a compound in which two or more atoms ofthe same molecule can coordinate with a metal to form a metal chelate.The two or more atoms may be, for example, oxygen, nitrogen, sulfur, andthe like. The atoms may be in the form of one or more functionalitiessuch as, for example, ketone, aldehyde, hydroxyl, amine, thioketone,thioaldehyde, thiol, and the like. The functionalities may be part of abenzyl group or a condensed aromatic ring system derived from, forexample, naphthalene, anthracene, phenanthrene, acridine and so forth.

One of the aforementioned metals is coordinated with one or morechelating agents, particle examples of which include, for example,2-(1′,1′,1′,2′,2′,3′,3′-heptafluoro-4′,6′-hexanedion-6′-yl)-naphthalene(NHA),4,4′-bis(2″,3″,3″-heptafluroro-4″,6″-hexanedion-6″-yl)-o-terphenyl(BHHT),4,4′-bis(1″,1″,1″,2″,2″,3″,3″-heptafluoro-4″,6″-hexanedion-6″-yl)-chlorosulfo-o-terphenyl(BHHCT), phenanthroline (phen) and phenanthroline-related compounds(derivatives of phenanthroline) such as, e.g., phenanthroline carboxylicacid, 4,7-diphenyl-1,10-phenanthroline (DPP), and the like,3-(2-thienoyl, 1,1,1-trifluoroacetone (TTA),thiophenetrifluorobutanedione (TTB), 3-naphthoyl-1,1,1-trifluoroacetone(NPPTA), naphthyltrifluorobutanedione (NTA), trioctyl phosphine oxide(TOPO), triphenyl phosphine oxide (TPPO).3-benzoyl-1,1,1-trifluoroacetone (BFTA),2,2-dimethyl-4-perfluorobutynoyl-3-butanone (fod), 2,2′-dipyridyl (bpy),salicylic acid, bipyridylcarboxylic acid, aza crown etherstrioctylphosphine oxide, aza cryptands, and so forth as well ascombinations of the above. As mentioned above, in some embodiments themetal in the metal chelate is at least hexacoordinated. The metalchelate will be uncharged; thus, the number of acidic groups provided bythe chelating agent will equal the oxidation state of the metal.Exemplary of particular metal chelates, by way of illustration and notlimitation, include Eu(BHHCT)₂DPP, Eu(TTA)₃DPP, Eu(NTA)₃DPP,Eu(NHA)₃DPP, Eu(BHHT)₂DPP, and metal chelates as discussed in U.S. Pat.No. 6,916,667 (for example, columns 5-9) and U.S. Patent Application No.20060270063 (column 3-4), the relevant disclosures of which areincorporated herein by reference, and so forth.

Many of the chelating agents and metal chelates are known in the art andmany are commercially available. In general, metal chelates can beprepared from a metal chelating agent by combining a metal chloride withthe desired ratio of molecules of metal chelating agent in an aqueousbuffered solvent and sufficient base to take up hydrochloric acid thatis produced during the reaction. For example, metal chelates can beprepared by a procedure such as that described by Shinha, A. P.,“Fluorescences and laser action in rare earth chelates,” SpectroscopyInorganic Chemistry, Vol 2, (1971), 255-288.

The chemiluminescent reagent may include a group or functionality thatimparts hydrophilicity or aqueous solubility, which increaseswettability of solids with water and the solubility in aqueous systemsof compounds to which it is bound. One or more of such functionality maybe present on the olefinic compound or on the metal chelate or both.Such functional group or functionality can be a substituent having 1 to50 or more atoms and can include a sulfonate, sulfate, phosphate,amidine, phosphonate, carboxylate, hydroxyl particularly polyols, amine,ether, amide, and the like. Illustrative functional groups arecarboxyalkyl, sulfonoxyalkyl, CONHOCH₂COOH, CO-(glucosamine), sugars,dextran, cyclodextrin, SO₂NHCH₂COOH, SO₃H, CONHCH₂CH₂SO₃H, PO₃H₂,OPO₃H₂, hydroxyl, carboxyl, ketone, and combinations thereof. Such agroup or functionality may be introduced into the chelating agent bymethods that are well-known in the art for introducing such groups orfunctionalities into compounds.

After preparation of the chemiluminescent reagent, the chemiluminescentreagent may be placed in a suitable medium for storage until used in anassay. In many embodiments the medium is an aqueous medium, usually anaqueous buffered medium. The aqueous medium may be solely water or mayinclude from 0.1 to about 40 volume percent of a cosolvent such as, forexample, an organic solvent, which may be an alcohol, ether, ester,amine, amide, and the like. The pH for the medium will usually be in therange of about 4 to about 11, or in the range of about 5 to about 10, orin the range of about 6.5 to about 9.5. Various buffers may be used toachieve the desired pH and maintain the pH. Illustrative buffers includeborate, phosphate, carbonate, tris, barbital, PIPES, HEPES, IVIES, ACES,MOPS, BICINE, and the like. The particular buffer employed is notcritical, but with a particular chemiluminescent reagent one or anotherbuffer may be preferred. In some embodiments, the medium in which thechemiluminescent reagent is stored is substantially similar to, or thesame as, the medium for an assay for an analyte where thechemiluminescent reagent is one of the assay reagents employed. Thesolubility of the chemiluminescent reagent in an aqueous medium at roomtemperature is, for example, at least 50%, or at least 55%, or at least60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%,or at least 85%, or at least 90%, or at least 95% (weight to volume).

In a specific embodiment of the above, by way of illustration and notlimitation, the chemiluminescent reagent comprises a thioxene andEu(BHHCT)₂DPP, both of which are covalently bound to BSA, to which abinding partner for an analyte is also bound.

A member of a specific binding pair (“sbp member”) for use in thepresent embodiments for non-covalent linking is one of two differentmolecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Thespecific binding pair for use in the present embodiments is selectedfrom the group consisting of (i) small molecule and binding partner forthe small molecule and (ii) large molecule and binding partner for thelarge molecule. In some embodiments, the small molecule has a molecularweight less than about 2000, or less than about 1500, or less than about1000, or less than about 500, or less than about 400, or less than about300, or the like. Examples of small molecule-binding partner for thesmall molecule pairs, by was of illustration and not limitation, includebiotin-binding partner for biotin (e.g., avidin, streptavidin, antibodyfor biotin, etc.), digoxin-binding partner for digoxin (e.g., antibodyfor digoxin, etc.), fluorescein-binding partner for fluorescein(antibody for fluorescein, etc.), rhodamine-binding partner forrhodamine (e.g., antibody for rhodamine), peptide-binding partner forthe peptide (antibody for the peptide, etc.), analyte-specific bindingpartners (e.g., intrinsic factor for B12, folate binding factor forfolate) and so forth.

In some embodiments of a specific binding pair for use in the presentembodiments, the molecular weight of the large molecule is greater thanabout 2,000, or greater than about 5,000, or greater than about 10,000,or greater than about 50,000, or greater than about 100,000, or greaterthan about 500,000, or greater than about 1,000,000, or greater thanabout 5,000,000 or greater than about 10,000,000, or the like. Examplesof large molecule-binding partner for the large molecule pairs, by wayof illustration and not limitation, include members of an immunologicalpair such as antigen-antibody, hormone-hormone receptors, nucleic acidduplexes, IgG-protein A, polynucleotide pairs such as DNA-DNA, DNA-RNA,other receptors and ligands, and the like.

The nature of the binding partner for the analyte is dependent primarilyon the nature of the analyte. The binding partner for the analyte may bean antibody, a polynucleotide, an analyte-specific binding protein otherthan an antibody, and so forth. An antibody is an immunoglobulin thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule. Theantibody can be monoclonal or polyclonal and can be prepared bytechniques that are well known in the art such as immunization of a hostand collection of sera (polyclonal) or by preparing continuous hybridcell lines and collecting the secreted protein (monoclonal), or bycloning and expressing nucleotide sequences or mutagenized versionsthereof coding at least for the amino acid sequences required forspecific binding of natural antibodies. Antibodies may include acomplete immunoglobulin or fragment thereof, which immunoglobulinsinclude the various classes and isotypes, such as IgA, IgD, IgE, IgG1,IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereof may include Fab, Fvand F(ab′)₂, Fab′, and the like. In addition, aggregates, polymers, andconjugates of immunoglobulins or their fragments can be used whereappropriate so long as binding affinity for a particular molecule ismaintained.

An analyte may be a ligand, which is monovalent (monoepitopic) orpolyvalent (polyepitopic), usually antigenic or haptenic, and is asingle compound or plurality of compounds which share at least onecommon epitopic or determinant site. The analyte can be a part of a cellsuch as a bacterium or a cell bearing a blood group antigen such as A,B, D, etc., or an HLA antigen or the analyte may be a microorganism,e.g., bacterium, fungus, protozoan, or virus.

The polyvalent ligand analytes may be poly(amino acids), i.e.,polypeptides and proteins, polysaccharides, nucleic acids, andcombinations thereof. Such combinations include components of bacteria,viruses, chromosomes, genes, mitochondria, nuclei, cell membranes andthe like. For the most part, the polyepitopic ligand has a molecularweight of at least about 5,000, more usually at least about 10,000. Inthe poly(amino acid) category, the poly(amino acids) of interest may befrom about 5,000 to 5,000,000 molecular weight, more usually from about20,000 to 1,000,000 molecular weight; among the hormones of interest,the molecular weights will usually range from about 5,000 to 60,000molecular weight.

The analyte may be a protein such as, for example, immunoglobulins,cytokines, enzymes, hormones, cancer antigens, nutritional markers,tissue specific antigens, etc. Such proteins include, by way ofillustration and not limitation, protamines, histones, albumins,globulins, scleroproteins, phosphoproteins, mucoproteins,chromoproteins, lipoproteins, nucleoproteins, glycoproteins, T-cellreceptors, proteoglycans, HLA, unclassified proteins, e.g.,somatotropin, prolactin, insulin, pepsin, proteins found in humanplasma, blood clotting factors, protein hormones such as, e.g.,follicle-stimulating hormone, luteinizing hormone, luteotropin,prolactin, chorionic gonadotropin, tissue hormones, cytokines, cancerantigens such as, e.g., PSA, CEA, a-fetoprotein, acid phosphatase,CA19.9, CA15.3 and CA125, tissue specific antigens, such as, e.g.,alkaline phosphatase, myoglobin, CK-MB and calcitonin, and peptidehormones. Other analytes of interest are mucopolysaccharides andpolysaccharides.

The monoepitopic ligand analytes will generally be from about 100 to2,000 molecular weight, more usually from about 125 to about 1,000molecular weight. The analytes include drugs (e.g., drugs of abuse,therapeutic drugs, etc.), metabolites, pesticides, pollutants, and thelike. Representative drugs of abuse (including misused drugs), by way ofexample and not limitation, include (i) alkaloids such as morphinealkaloids, which include morphine, codeine, heroin, dextromethorphan,their derivatives and metabolites; cocaine alkaloids, which includecocaine and benzyl ecgonine, their derivatives and metabolites; ergotalkaloids, which include the diethylamide of lysergic acid; steroidalkaloids; iminazoyl alkaloids; quinazoline alkaloids; isoquinolinealkaloids; quinoline alkaloids, which include quinine and quinidine;diterpene alkaloids, their derivatives and metabolites; (ii) steroids,which include the estrogens, androgens, andreocortical steroids, bileacids, cardiotonic glycosides and aglycones, which includes digoxin anddigoxigenin, saponins and sapogenins, their derivatives and metabolites;steroid mimetic substances, such as diethylstilbestrol; (iii) lactamshaving from 5 to 6 annular members, which include the barbiturates,e.g., phenobarbital and secobarbital, diphenylhydantoin, primidone,ethosuximide, and their metabolites; (iv) aminoalkylbenzenes, with alkylchain of from 2 to 3 carbon atoms, which include the amphetamines;catecholamines, which include ephedrine, L-dopa, epinephrine; narceine;papaverine; and metabolites of the above; (v) benzheterocyclics whichinclude oxazepam, chlorpromazine, tegretol, their derivatives andmetabolites, the heterocyclic rings being azepines, diazepines andphenothiazines; (vi) purines, which includes theophylline, caffeine,their metabolites and derivatives; (vii) drugs derived from marijuana,which include cannabinol and tetrahydrocannabinol; (viii) hormones suchas thyroxine, cortisol, triiodothyronine, testosterone, estradiol,estrone, progesterone, (ix) tricyclic antidepressants, which includeimipramine, desmethylimipramine, amitriptyline, nortriptyline,protriptyline, trimipramine, chlomipramine, doxepine, anddesmethyldoxepin; and (x) anti-neoplastics, which include methotrexate;and the like.

General Description of Assays for an Analyte Utilizing the PresentReagents

Embodiments of the present invention have application to assays for thedetermination of an analyte. In general, in such assays the reagentscomprise, among others, a binding partner for the analyte. A samplesuspected of containing an analyte is combined in an assay medium with abinding partner for the analyte. A determination is made of the extentof binding between the analyte and the binding partner for the analyte.A chemiluminescent reagent in accordance with the present embodiments isemployed as a label reagent in the detection of this binding event. Theassay can be performed either without separation (homogeneous) or withseparation (heterogeneous) of any of the assay components or products.Heterogeneous assays usually involve one or more separation steps andcan be competitive or non-competitive.

Some known assays utilize a signal producing system (sps) that employs aparticulate chemiluminescent reagent, which has a metal chelateassociated with a support and has at least first and second sps members.The designation “first” and “second” is completely arbitrary and is notmeant to suggest any order or ranking among the sps members or any orderof addition of the sps members in the present methods. The sps membersmay be related in that activation of one member of the sps produces aproduct such as, e.g., light, which results in activation of anothermember of the sps. In some embodiments of known assays, the sps memberscomprise a sensitizer and a chemiluminescent composition whereactivation of the sensitizer results in a product that activates thechemiluminescent composition. The second sps member usually generates adetectable signal that relates to the amount of bound and/or unbound spsmember, i.e. the amount of sps member bound or not bound to the analytebeing detected or to an agent that reflects the amount of the analyte tobe detected. In accordance with the present invention, thenon-particulate chemiluminescent reagent in accordance with the presentembodiments and described above may be employed in place of theparticulate chemiluminescent reagent of the known methods.

In some embodiments of methods in accordance with the presentembodiments, the first sps member is a sensitizer, such as, for example,a photosensitizer and the second sps member is the non-particulatechemiluminescent reagent of the present embodiments that is activated asa result of the activation of the first sps member. The sensitizer maybe any moiety that upon activation produces a product that activates thechemiluminescent reagent, which in turn generates a detectable signal.In many embodiments the sensitizer is capable of generating singletoxygen upon activation.

In some embodiments the sensitizer is a photosensitizer for generationof singlet oxygen usually by excitation with light. The photosensitizercan be photoactivatable (e.g., dyes and aromatic compounds) orchemi-activated (e.g., enzymes and metal salts). When excited by lightthe photosensitizer is usually a compound comprised of covalently bondedatoms, usually with multiple conjugated double or triple bonds. Thecompound should absorb light in the wavelength range of about 200 toabout 1100 nm, or about 300 to about 1000 nm, or about 450 to about 950nm, with an extinction coefficient at its absorbance maximum greaterthan about 500 M⁻¹ cm⁻¹, or at least about 5000 M⁻¹ cm⁻¹, or at leastabout 50,000 M⁻¹ cm⁻¹ at the excitation wavelength. Photosensitizersthat are to be excited by light will be relatively photostable and willnot react efficiently with singlet oxygen. Several structural featuresare present in most useful photosensitizers. Most photosensitizers haveat least one and frequently three or more conjugated double or triplebonds held in a rigid, frequently aromatic structure. Thephotosensitizer usually contains at least one group that acceleratesintersystem crossing such as a carbonyl or imine group or a heavy atomselected from rows 3-6 of the periodic table, especially iodine orbromine, or they may have extended aromatic structures. Typicalphotosensitizers include acetone, benzophenone, 9-thioxanthone, eosin,9,10-dibromoanthracene, methylene blue, metallo-porphyrins, such ashematoporphyrin, phthalocyanines, chlorophylis, rose bengal,buckminsterfullerene, etc., and derivatives of these compounds havingsubstituents of 1 to 50 atoms for rendering such compounds morelipophilic or more hydrophilic and/or as attaching groups forattachment, for example, to an sps member or an sbp member.

The photosensitizers useful in the above methods include othersubstances and compositions that can produce singlet oxygen with or,less preferably, without activation by an external light source. Thus,for example, molybdate salts and chloroperoxidase and myeloperoxidaseplus bromide or chloride ion (Kanofsky, J. Biol. Chem. (1983) 259 5596)have been shown to catalyze the conversion of hydrogen peroxide tosinglet oxygen and water. Also included within the scope ofphotosensitizers are compounds that are not true sensitizers but whichon excitation by heat, light, or chemical activation will release amolecule of singlet oxygen. The best known members of this class ofcompounds includes the endoperoxides such as1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen. Examples of otherphotosensitizers that may be utilized are those set forth in U.S. Pat.Nos. 5,340,716 and 6,251,581, the relevant disclosures of which areincorporated herein by reference.

In a particular embodiment, the present invention has application in theinduced luminescence immunoassay referred to in U.S. Pat. No. 5,340,716(Ullman) entitled “Assay Method Utilizing PhotoactivatedChemiluminescent Label” (“induced luminescence assay”), which disclosureis incorporated herein by reference. In one approach in accordance withthe present embodiments, the assay uses a particle incorporating aphotosensitizer and a non-particulate chemiluminescent reagent asdescribed above. The binding partner for an analyte of the presentchemiluminescent reagent binds to an analyte to form a complex, or bindsto a second sbp member to form a complex, in relation to the presence ofthe analyte. If the analyte is present, the photosensitizer and thechemiluminescent compound come into close proximity by virtue of thebinding, to the analyte, of the binding partner for the analyte on thephotosensitizer particle and the binding partner for the analyte that ispart of the non-particulate chemiluminescent reagent in accordance withthe present embodiments. The photosensitizer generates singlet oxygenand activates the non-particulate chemiluminescent reagent when the twolabels are in close proximity. The activated chemiluminescent reagentsubsequently produces light. The amount of light produced is related tothe amount of the complex formed, which in turn is related to the amountof analyte present.

In some embodiments of the induced luminescence assay, a photosensitizerparticle is employed that is conjugated to avidin. A biotinylatedbinding partner for an analyte is also employed. A non-particulatechemiluminescent reagent in accordance with the present embodiments isemployed as part of the detection system. The reaction medium isincubated to allow the photosensitizer particles to bind to thebiotinylated binding partner for the analyte by virtue of the bindingbetween avidin and biotin and to also allow the binding partner for theanalyte that is part of the photosensitizer reagent and the bindingpartner for the analyte that is part of the non-particulatechemiluminescent reagent in accordance with the present embodiments tobind to the analyte. Then, the medium is irradiated with light to excitethe photosensitizer, which is capable in its excited state of activatingoxygen to a singlet state. Because the chemiluminescent reagent is nowin close proximity to the photosensitizer by virtue of the presence ofthe analyte, it is activated by the singlet oxygen and emitsluminescence. The medium is then examined for the presence and/or theamount of luminescence or light emitted, the presence thereof beingrelated to the presence and/or amount of the analyte.

The sample to be analyzed is one that is suspected of containing ananalyte. The samples are preferably from humans or animals and includebiological fluids such as whole blood, serum, plasma, sputum, lymphaticfluid, semen, vaginal mucus, feces, urine, spinal fluid, saliva, stool,cerebral spinal fluid, tears, mucus, and the like; biological tissuesuch as hair, skin, sections or excised tissues from organs or otherbody parts; and so forth. In many instances, the sample is whole blood,plasma or serum.

The sample can be prepared in any convenient medium. Conveniently, thesample may be prepared in an assay medium, which is discussed more fullyherein. In some instances a pretreatment may be applied to the samplesuch as, for example, to lyse blood cells, and the like. Suchpretreatment is usually performed in a medium that does not interferesubsequently with an assay. An aqueous medium is preferred for thepretreatment.

As discussed briefly above, the assays are normally carried out in anaqueous buffered medium at a moderate pH, generally that which providesoptimum assay sensitivity. The aqueous medium may be solely water or mayinclude from 0.1 to about 40 volume percent of a cosolvent. The pH forthe medium will usually be in the range of about 4 to about 11, moreusually in the range of about 5 to about 10, and preferably in the rangeof about 6.5 to about 9.5. The pH will usually be a compromise betweenoptimum binding of the binding members of any specific binding pairs,the pH optimum for other reagents of the assay such as members of thesignal producing system, and so forth. Various buffers may be used toachieve the desired pH and maintain the pH during the determination.Illustrative buffers include borate, phosphate, carbonate, tris,barbital, PIPES, HEPES, IVIES, ACES, MOPS, BICINE, and the like. Theparticular buffer employed is not critical, but in an individual assayone or another buffer may be preferred.

Various ancillary materials may be employed in the above methods. Forexample, in addition to buffers the medium may comprise stabilizers forthe medium and for the reagents employed. In some embodiments, inaddition to these additives, proteins may be included, such as albumins;organic solvents such as formamide; quaternary ammonium salts;polyanions such as dextran sulfate; binding enhancers, e.g.,polyalkylene glycols; polysaccharides such as dextran, trehalose, or thelike. The medium may also comprise agents for preventing the formationof blood clots. Such agents are well known in the art and include, forexample, EDTA, EGTA, citrate, heparin, and the like. The medium may alsocomprise one or more preservatives as are known in the art such as, forexample, sodium azide, neomycin sulfate, PROCLIN® 300, Streptomycin, andthe like. Any of the above materials, if employed, is present in aconcentration or amount sufficient to achieve the desired effect orfunction.

One or more incubation periods may be applied to the medium at one ormore intervals including any intervals between additions of variousreagents mentioned above. The medium is usually incubated at atemperature and for a time sufficient for binding of various componentsof the reagents to occur. Moderate temperatures are normally employedfor carrying out the method and usually constant temperature,preferably, room temperature, during the period of the measurement.Incubation temperatures normally range from about 5° to about 99° C.,usually from about 15° C. to about 70° C., more usually 20° C. to about45° C. The time period for the incubation is about 0.2 seconds to about24 hours, or about 1 second to about 6 hours, or about 2 seconds toabout 1 hour, or about 1 to about 15 minutes. The time period depends onthe temperature of the medium and the rate of binding of the variousreagents, which is determined by the association rate constant, theconcentration, the binding constant and dissociation rate constant.Temperatures during measurements will generally range from about 10 toabout 50° C., or from about 15 to about 40° C.

The concentration of the analyte that may be assayed generally variesfrom about 10⁻⁵ to about 10⁻¹⁷ M, more usually from about 10⁻⁶ to about10⁻¹⁴ M. Considerations, such as whether the assay is qualitative,semi-quantitative or quantitative (relative to the amount of the analytepresent in the sample), the particular detection technique and theconcentration of the analyte normally determine the concentrations ofthe various reagents.

The concentrations of the various reagents in the assay medium willgenerally be determined by the concentration range of interest of theanalyte, the nature of the assay, and the like. However, the finalconcentration of each of the reagents is normally determined empiricallyto optimize the sensitivity of the assay over the range. That is, avariation in concentration of analyte that is of significance shouldprovide an accurately measurable signal difference. Considerations suchas the nature of the signal producing system and the nature of theanalytes normally determine the concentrations of the various reagents.

As mentioned above, the sample and reagents are provided in combinationin the medium. While the order of addition to the medium may be varied,there will be certain preferences for some embodiments of the assayformats described herein. The simplest order of addition, of course, isto add all the materials simultaneously and determine the effect thatthe assay medium has on the signal as in a homogeneous assay.Alternatively, each of the reagents, or groups of reagents, can becombined sequentially. In some embodiments, an incubation step may beinvolved subsequent to each addition as discussed above.

As mentioned above, embodiments of the aforementioned assays employ aparticulate photosensitizer reagent. The particle may be comprised of anorganic or inorganic, solid or fluid, water insoluble material, whichmay be transparent or partially transparent. The particles generallyhave an average diameter of at least about 0.02 microns and not morethan about 100 microns. In some embodiments, the particles have anaverage diameter from about 0.05 microns to about 20 microns, or fromabout 0.3 microns to about 10 microns. The particle may be organic orinorganic, swellable or non-swellable, porous or non-porous, preferablyof a density approximating water, generally from about 0.7 g/mL to about1.5 g/mL, and composed of material that can be transparent, partiallytransparent, or opaque. The particles can be biological materials suchas cells and microorganisms, e.g., erythrocytes, leukocytes,lymphocytes, hybridomas, streptococcus, Staphylococcus aureus, E. coli,viruses, and the like. The particles can also be particles comprised oforganic and inorganic polymers, liposomes, latex particles, magnetic ornon-magnetic particles, phospholipid vesicles, chylomicrons,lipoproteins, and the like. In some embodiments, the particles arechrome particles or latex particles.

Examination Step

In a next step of an assay method, the medium is examined for thepresence of a complex comprising the analyte and the binding partner forthe analyte. The presence and/or amount of the complex indicates thepresence and/or amount of the analyte in the sample.

The phrase “measuring the amount of an analyte” refers to thequantitative, semiquantitative and qualitative determination of theanalyte. Methods that are quantitative, semiquantitative andqualitative, as well as all other methods for determining the analyte,are considered to be methods of measuring the amount of the analyte. Forexample, a method, which merely detects the presence or absence of theanalyte in a sample suspected of containing the analyte, is consideredto be included within the scope of the present invention. The terms“detecting” and “determining,” as well as other common synonyms formeasuring, are contemplated within the scope of the present invention.

In many embodiments the examination of the medium involves detection ofa signal from the medium where the signal produced results from theinvolvement of the chemiluminescent composition in accordance with thepresent embodiments. The presence and/or amount of the signal is relatedto the presence and/or amount of the analyte in the sample. Theparticular mode of detection depends on the nature of the sps. Asdiscussed above, there are numerous methods by which a label of an spscan produce a signal detectable by external means. Activation of asignal producing system depends on the nature of the signal producingsystem members. For an sps member that is a sensitizer that is activatedby light, the sps member is irradiated with light. Other activationmethods will be suggested to those skilled in the art in view of thedisclosures herein.

When a photosensitizer is used, the photosensitizer serves to activatethe chemiluminescent reagent when the medium containing the abovereactants is irradiated. The medium is irradiated with light having awavelength of sufficient energy to convert the photosensitizer to anexcited state and render it capable of activating molecular oxygen tosinglet oxygen. When bound to a binding partner for the analyte, thephotosensitizer concentration may be very low, frequently about 10⁻⁶ toabout 10⁻¹² M or lower. Generally, for the above embodiments involving aphotosensitizer, the medium is irradiated with light having a wavelengthof about 300 to about 1200 nm, or about 450 to about 950, or about 550to about 800 nm.

The period of irradiation will depend on the lifetime of the activatedchemiluminescent composition of embodiments of the presentchemiluminescent reagents and the light intensity and the desiredemission intensity. For short-lived activated chemiluminescentcompositions, the period may be less than a second, usually about amillisecond but may be as short as a microsecond where an intenseflashlamp or laser is used. For longer-lived activated chemiluminescentcompositions, the irradiation period can be longer and a less intensesteady light source can be used. In general, the integrated lightintensity over the period of irradiation should be sufficient to exciteat least 0.1% of the photosensitizer molecules, preferably at least 30%,and, most preferably, every photosensitizer molecule will be excited atleast once.

A helium-neon laser is an inexpensive light source for excitation at632.6 nm. Photosensitizers that absorb light at this wavelength arecompatible with the emission line of a helium-neon laser and are,therefore, particularly useful in the present methods in whichphotosensitizers are employed. Other light sources include, for example,other lasers such as Argon, YAG, He/Cd, and ruby; photodiodes; mercury,sodium and xenon vapor lamps; incandescent lamps such as tungsten andtungsten/halogen; and flashlamps.

Temperatures during measurements generally range from about 10° to about70° C., or from about 20° to about 45° C., or about 20° to about 25° C.In one approach standard curves are formed using known concentrations ofthe analytes to be screened. As discussed above, calibrators and othercontrols may also be used.

The luminescence or light produced in any of the above approaches can bemeasured visually, photographically, actinometrically,spectrophotometrically or by any other convenient means to determine theamount thereof, which is related to the amount of analyte in the medium.The examination for presence and/or amount of the signal also includesthe detection of the signal, which is generally merely a step in whichthe signal is read. The signal is normally read using an instrument, thenature of which depends on the nature of the signal. The instrument maybe a spectrophotometer, fluorometer, absorption spectrometer,luminometer, chemiluminometer, and the like. The presence and amount ofsignal detected is related to the presence and amount of the analytepresent in a sample.

A helium-neon laser is an inexpensive light source for excitation at632.6 nm. Photosensitizers that absorb light at this wavelength arecompatible with the emission line of a helium-neon laser and are,therefore, particularly useful in the present methods in whichphotosensitizers are employed. Other light sources include, for example,other lasers such as Argon, YAG, He/Cd, and ruby; photodiodes; mercury,sodium and xenon vapor lamps; incandescent lamps such as tungsten andtungsten/halogen; and flashlamps.

Kits Comprising Reagents for Conducting Assays

The present chemiluminescent reagent and other reagents for conducting aparticular assay may be present in a kit useful for convenientlyperforming an assay for the determination of an analyte. In someembodiments a kit comprises in packaged combination a biotin-bindingpartner for analyte conjugate, streptavidin-sensitizer particles and anon-particulate chemiluminescent reagent in accordance with the presentembodiments wherein the binding partner for the analyte of thechemiluminescent reagent recognizes and binds to a different epitope onthe analyte than the binding partner for the analyte that is part of thebiotin-binding partner for the analyte conjugate. The kit may furtherinclude other reagents for performing the assay, the nature of whichdepend upon the particular assay format.

The reagents may each be in separate containers or various reagents canbe combined in one or more containers depending on the cross-reactivityand stability of the reagents. The kit can further include otherseparately packaged reagents for conducting an assay such as additionalsbp members, ancillary reagents, and so forth.

The relative amounts of the various reagents in the kits can be variedwidely to provide for concentrations of the reagents that substantiallyoptimize the reactions that need to occur during the present method andfurther to optimize substantially the sensitivity of the assay. Underappropriate circumstances one or more of the reagents in the kit can beprovided as a dry powder, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentrations for performing a method or assay inaccordance with the present invention. The kit can further include awritten description of a method in accordance with the present inventionas described above.

The phrase “at least” as used herein means that the number of specifieditems may be equal to or greater than the number recited. The phrase“about” as used herein means that the number recited may differ by plusor minus 10%; for example, “about 5” means a range of 4.5 to 5.5. Thedesignations “first” and “second” are used solely for the purpose ofdifferentiating between two items such as, for example, “first spsmember” and “second sps member,” and are not meant to imply any sequenceor order or importance to one item over another.

The following examples further describe the specific embodiments of theinvention by way of illustration and not limitation and are intended todescribe and not to limit the scope of the invention. Parts andpercentages disclosed herein are by volume unless otherwise indicated.

EXAMPLES Materials

Testing was conducted using the DIMENSION® RxL analyzer, available fromSiemens Healthcare Diagnostics Inc., Newark Del. The instrument wasemployed using induced luminescence immunoassay technology and wasequipped with an appropriate reader.

Unless indicated otherwise, reagents were purchased from Sigma-Aldrich(Milwaukee, Wis.) and used as received unless mentioned otherwise. BHHCTwas synthesized as described by Yuan J. and Matsumoto K. (Anal. Biochem.1998, 70: 596-601). Anti-TSH antibody, a monoclonal antibody, wasprepared by known somatic cell hybridization techniques; see, forexample, Köhler and Milstein, Nature 265:495-497, 1975.

Purity of samples was analyzed by analytical thin layer chromatography(TLC) performed on Analtech Uniplate Silica Gel GF (0.25 mm)glass-backed plates using the specified solvent. TLC spots werevisualized by ultraviolet light (short and/or long wave length) and/oriodine vapors. Flash chromatography was carried out on Whatman silicagel 60 Å (230-400 mesh). ¹H-NMR spectra were recorded on a BrukerUltrashiel™-400 (400 MHz) spectrometer. Chemical shifts are reported inparts per million (ppm, δ) relative to tetramethylsilane as internalreference. NMR abbreviations used are s (singlet), d (doublet), m(multiplet) and t (triplet).

Preparation of Compound II (FIG. 1):

N-methyl aniline (I) (25 g, 233.3 mmol) and ethyl 5-bromo-valerate (25g, 119.6 mmol) were added to a 100 ml round bottom flask. The contentsof the flask were heated with stirring at 100° C. for 16 hours. Thereaction mixture was allowed to cool to room temperature and was pouredinto ethyl acetate (50 mL). The ethyl acetate solution was washed with20% sodium hydroxide (3×50 mL); the combined sodium hydroxide solutionwas extracted back once with ethyl acetate (25 mL) and the remainingaqueous sodium hydroxide solution was discarded. The combined ethylacetate extract was washed with water (10 mL) and dried over MgSO₄. Theclear ethyl acetate solution was concentrated to dryness on a rotaryevaporator. Residual oil so obtained was purified by distillation underhigh vacuum (130-134° C., 0.5 mm Hg) to yield 21 g of a colorless liquidcompound II. ¹H-NMR (CDCl₃) δ: 7.18 (m, 2H), 6.68 (m, 3H), 4.2 (q, J=8.0Hz, 2H), 3.36 (t, J=8.0 Hz, 2H), 2.92 (s, 3H), 2.33 (t, J=8.0 Hz, 2H),1.64 (m, 4H), 1.24 (t, J=8.0 Hz, 3H).

Preparation of Compound III (FIG. 1):

Phosphorus oxychloride (POCl₃; 5 g, 33 mmol) was added dropwise over aperiod of 10 minutes, through a dropping funnel, to stirred N,N-dimethylformamide (DMF; 8.8 g, 120 mmol) at 0-4° C. After 10 minutes at 0-4° C.,compound II (3.7 g, 15.96 mmol) was added rapidly in one portion. Thevial used for compound II was rinsed with 1 ml of DMF and the DMFsolution was added to the stirred reaction mixture, which was heatedwith stirring at 100° C. for 1 hour. The reaction mixture was allowed tocool to room temperature and poured slowly into an ice-water mixture.The aqueous phase was neutralized with 20% sodium hydroxide andextracted with ethyl acetate (3×50 mL). The combined ethyl acetatesolution was washed with water (20 mL) and dried over MgSO₄. The clearorganic solution was concentrated to dryness under reduced pressure. Theresulting oily residue was purified by flash column chromatography usingethyl acetate/dichloromethane (9/1; v/v) to give 2.46 g of the compoundIII ¹H-NMR (CDCl₃) δ: 9.72 (s, 1H), 7.18 (d, J=8.0 Hz, 2H), 6.68 (d,J=8.0 Hz, 2H), 4.22 (q, J=8.0 Hz, 2H), 3.40 (m, 2H), 3.05 (s, 3H), 2.35(t, J=8.0 Hz, 2H), 1.65 (m, 4H), 1.25 (t, J=8.0 Hz, 3H).

Preparation of Compound IV (FIG. 1):

Potassium cyanide (1.0 g, 15.3 mmol) was added to a solution of compoundIII (2.46 g, 9.34 mmol) in 15 mL of 60% ethanol stirred in a nitrogenatmosphere. The mixture was heated under reflux for 15 minutes.Benzaldehyde (1.05 g) dissolved in 10 ml ethanol was added to therefluxed reaction mixture over a period of 45 minutes. After 15 minutes,the cooled reaction mixture was poured into ethyl acetate (30 mL). Theaqueous phase was separated and extracted back with ethyl acetate (3×30mL). The combined ethyl acetate solution was dried over MgSO₄ and theclear solution was concentrated to dryness under reduced pressure. Crudereaction mixture was dissolved in 10 ml of dry ethanol, mixed with 0.25ml of trimethylsilyl chloride and stirred for 16 hr at room temperature.The reaction mixture was poured into a mixture of saturated sodiumbicarbonate (120 mL) and dichloromethane (30 mL). The aqueous phase wasseparated and extracted with dichloromethane (2×30 mL). The combinedorganic phase was washed with saturated sodium bicarbonate (60 ml) anddried over MgSO₄. The clear organic solution was concentrated to drynessunder reduced pressure. The oily residue was purified by flash columnchromatography using ethyl acetate/hexane (1/5; v/v) to give 196 mg ofthe compound IV. ¹H-NMR (CDCl₃) δ: 7.84 (d, J=8 Hz, 2H), 7.34 (m, 5H),6.54 (d, J=8 Hz, 2H), 5.86 (d, J=4 Hz, 1H), 4.86 (d, J=4 Hz, 1H), 4.14(q, J=8.0 Hz, 2H), 3.38 (t, J=8.0 Hz, 2H), 3.00 (s, 3H), 2.34 (t, J=8.0Hz, 2H), 1.64 (m, 4H), 1.25 (t, J=8.0 Hz, 3H).

Preparation of Compound V (FIG. 1):

A stirred solution of compound IV (176 mg, 0.476 mmol) in 8 mL of drytoluene was mixed with thioethanol (0.28 ml, 312 mg, 4.0 mmol) andtrimethylsilyl chloride (0.51 mL, 436 mg, 4.0 mmol). After heating underreflux for 24 hr, under nitrogen, the cooled reaction mixture was pouredinto saturated sodium bicarbonate (15 mL). The organic phase wasseparated and washed with saturated sodium bicarbonate (10 mL). Thecombined aqueous phase was extracted back with dichloromethane (3×30 mL)and the dichloromethane extract washed with water (20 mL). The combineddichloromethane solution was dried over MgSO₄, filtered and concentratedto dryness. The oily residue was purified by preparative thin layerchromatography using ethyl acetate/dichloromethane (5/95; v/v) to give93 mg of the compound V. ¹H-NMR (CDCl₃) δ: 7.20-7.04 (m, 7H), 6.50 (d,J=8.0 Hz, 2H), 4.48 (m, 2H), 4.12 (q, J=8.0 Hz, 2H), 3.28 (m, 2H), 3.20(m, 2H), 2.87 (s, 3H), 2.32 (t, J=8.0 Hz, 2H), 1.60 (m, 4H), 1.25 (t,J=8.0 Hz, 3H).

Preparation of Compound VI (FIG. 1):

Sodium hydroxide (30 mg, 0.75 mmol) was added to a stirred solution ofcompound V (28 mg, 0.068 mmol) in MeOH (1 mL)-THF (2 ml)-water (0.5 mL).The reaction mixture was stirred at room temperature for 4 hr when TLCanalysis showed absence of any starting product. The reaction mixturewas mixed with 1.5 mL of water and the pH was adjusted to 3.0 by a slowaddition of 1 N HCl. The reaction mixture was evaporated to drynessunder vacuum. The oily residue was dissolved in dichloromethane (50 ml)and washed with 10 mL of water. The clear organic solution was driedover MgSO₄, filtered and concentrated to dryness to give 23.6 mg of thecompound VI. FAB-MS (negative mode) m/z: 382 [(M-H)⁻, 100); ¹H-NMR(CD₃OD) δ: 7.15-7.09 (m, 7H), 6.81 (d, J=8.0 Hz, 2H), 4.47 (m, 2H), 3.35(m, 2H), 3.22 (m, 2H), 2.97 (s, 3H), 2.31 (t, J=8.0 Hz, 2H), 1.58 (m,4H).

Synthesis of BSA-Coupled Thioxene Carboxylate:

The thioxene carboxylate VI (12.2 mg; 32 mmol) was dissolved in 0.5 mLDMF. The mixture was mixed with a 0.1 mL DMF solution ofN-hydroxysuccinimide (NETS) (16 mg; 140 mmol) and a 0.1 mL DMF solutionof dicyclohexyl carbodiimide (16 mg; 79 mmol). After stirring for 16 hrat ambient temperature, the organic reaction mixture was added slowly toa 5-mL solution of bovine serum albumin (BSA) (55 mg; 0.82 mmol) in 100mM sodium phosphate-0.2 mM europium chloride, pH 7.60. After 4 hr,precipitated solid was separated by centrifugation and discarded. Theclear solution was passed through a Sephadex G25 column (2.6×30 cm)equilibrated and eluted with 100 mM sodium bicarbonate-0.2 mM europiumchloride, pH 8.20. Protein-containing fractions were combined.BSA-thioxene, thus obtained, was calculated to incorporate 31 thioxeneresidues per mole of the protein utilizing an extinction coefficient of1.33×10⁴ mole⁻¹ cm⁻¹ at 330 nm.

Preparation of Activated BSA-Thioxene:

A 12 mL solution of BSA-thioxene (2 mg/mL protein; 0.36 mmol) was mixedwith 1.2 mL of a 10 mg/mL solution of sulfoSMPB (sulfosuccinimidyl[4-[-maleimidophenyl]butyrate]); 26.2 mmol). After 3 hr at ambienttemperature, the protein solution was separated from excess reagents bypassage through a Sephadex G25 column (2.6×30 cm) equilibrated andeluted with 100 mM sodium phosphate-200 μM europium chloride, pH 6.0.

Preparation of IgG-BSA-Thioxene:

Coupling of activated BSA-thioxene from above with reduced antibody wascarried out by mixing a 3.0 mL solution of anti-TSH antibody (referredto as “IgG” in above conjugate) with 0.33 mL of a 100 mM solution ofdithiothreitol in 100 mM sodium phosphate-5.0 mM EDTA, pH 6.0. Afterheating at 37° C. for one hour, the protein solution was passed througha Sephadex G25 column (1.6×30 cm) equilibrated and eluted with 100 mMsodium phosphate-200 μM europium chloride, pH 6.0. The reduced antibodywas calculated to contain 7.2 moles of free sulfhydryls per mole of theprotein. A 7.3 mL solution of this reduced antibody containing 19 mgprotein (136 μmoles) was mixed with 28 mL of activated BSA-thioxenecontaining 31 mg protein (463 μmoles) and the reaction mixtureconcentrated to 3 mL after adjusting pH to 7.0. After 16 hr at 4° C.,the reaction mixture was quenched with 50 μL of a 20 mg/mL aqueoussolution of N-ethylmaleimide. The protein solution was then purified bygel filtration over an AcA-34 column (1.6×70 cm) equilibrated and elutedwith 50 mM Hepes-300 mM NaCl-0.2% polyethyleneglycol (PEG8000)-0.2 mMeuropium chloride, pH 8.0. Fractions, corresponding to first proteinpeak eluted from the column, were pooled and stored in presence of 0.25mg/mL of neomycin sulfate.

Reaction of IgG-BSA-Thioxene with BHHCT:

A 5 mL solution of the IgG-BSA-thioxene from above, containing about 1mg/mL protein, was mixed with a 0.5 mL DMF solution of BHHCT containing4.5 mg BHHCT. Reaction mixture was shaken at ambient temperature for 16hr and centrifuged at 3000 rpm for 10 min, and the clear solution waspassed through a Sephadex G50 column (2.6×30 cm) equilibrated and elutedwith 50 mM Hepes-300 mM NaCl-0.2% polyethyleneglycol (PEG8000)-0.2 mMeuropium chloride, pH 8.0. Protein-containing fractions were pooled andstored in presence of 0.25 mg/mL neomycin sulfate to giveIgG-BSA-(thioxene)(BHHCT).

Assay Utilizing IgG-BSA-(Thioxene)(BHHCT):

IgG-BSA-(thioxene)-(BHHCT) from above was diluted 1:100 in 50 mM HEPESbuffer containing 0.2% PEG8000, 2 mg/mL BSA, 50 μM EuCl₃, 1 μM DPP,0.15% PROCLIN300® and 0.2 mg/mL neomycin sulfate. In this manner, theBHHCT of the above reagent formed Eu(BHHCT)₂DPP in situ. The contents ofwells 5-8 of a Dimension Vista® TSH FLEX® (Cat No. K6412, Lot 06229AB)were removed and the wells were rinsed with deionized water andre-filled with diluted reagent (IgG-BSA-(thioxene)-(Eu(BHHCT)₂DPP) fromabove. Wells containing biotinylated anti-TSH antibody (R1, wells 1-4)and streptavidin Sensibeads (photosensitizer beads) (R3, wells 9-12)were left intact. The 6-level LOCI® 1 Calibrator (Cat No. KC660, Lot6BD036) was tested on VISTA® using this FLEX® and the VISTA® TSH methodparameters (VISTA® Software 2.0). The calibration curve is shown in FIG.2 wherein kcounts is kilocounts and TSH is thyroid stimulating hormone.The separation in LOCI® signal was 2.2-fold between level B and A, and101-fold between level F and A. The N=3 within-run % CV ranged from 1.1%to 6.3% for calibrators B to F. The results are shown in FIG. 3.

The FLEX® with the 1:100 diluted (IgG-BSA-(thioxene)-(Eu(BHHCT)₂DPP) wasstored at 2-8° C. and tested periodically using LOCI® 1 Calibrator. Overa period of 16 days, the LOCI® signal for all non-zero calibrators (B toF) were within 10% of the day 0 value. The results are shown in FIGS. 4and 5.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. Furthermore, the foregoing description,for purposes of explanation, used specific nomenclature to provide athorough understanding of the invention. However, it will be apparent toone skilled in the art that the specific details are not required inorder to practice the invention. Thus, the foregoing descriptions ofspecific embodiments of the present invention are presented for purposesof illustration and description; they are not intended to be exhaustiveor to limit the invention to the precise forms disclosed. Manymodifications and variations are possible in view of the aboveteachings. The embodiments were chosen and described in order to explainthe principles of the invention and its practical applications and tothereby enable others skilled in the art to utilize the invention.

1. A chemiluminescent reagent for determining the presence and/or amountof an analyte in a sample suspected of containing the analyte, thereagent being non-particulate and comprising a binding partner for theanalyte and a chemiluminescent composition comprising an olefiniccompound and a metal chelate.
 2. The chemiluminescent reagent accordingto claim 1 wherein the binding partner for the analyte is covalentlybound to the chemiluminescent composition by a bond or a linking group.3. The chemiluminescent reagent according to claim 2 wherein the bindingpartner for the analyte is covalently bound to the chemiluminescentcomposition by a linking group and wherein the linking group comprises ahydrophilic macromolecule.
 4. The chemiluminescent reagent according toclaim 3 wherein the olefinic compound and the metal chelate are linkedto the same molecule of the linking group to which the binding partnerfor the analyte is covalently bound.
 5. The chemiluminescent reagentaccording to claim 3 wherein the olefinic compound and the metal chelateare linked to separate molecules of the linking group to which thebinding partner for the analyte is covalently bound and wherein thelinking group may be the same or different.
 6. The chemiluminescentreagent according to claim 3 wherein the hydrophilic macromolecule isselected from the group consisting of polypeptides, dendrimers,polycarboxylates, polyamines, polysulfhydryls and polyethyleneglycols.7. The chemiluminescent reagent according to claim 1 wherein the bindingpartner for the analyte is non-covalently bound to the chemiluminescentcomposition by a specific binding pair wherein one member of thespecific binding pair is linked to the binding partner for the analyteand the other member of the specific binding pair is linked to thechemiluminescent composition by a bond or a linking group.
 8. Thechemiluminescent reagent according to claim 7 wherein the bindingpartner for the analyte is non-covalently bound to the chemiluminescentcomposition by a specific binding pair wherein one member of thespecific binding pair is linked to the binding partner for the analyteand the other member of the specific binding pair is linked to thechemiluminescent composition by a linking group wherein the linkinggroup is a protein.
 9. The chemiluminescent reagent according to claim 8wherein the olefinic compound and the metal chelate are linked to thesame molecule of the linking group to which the binding partner for theanalyte is non-covalently bound.
 10. The chemiluminescent reagentaccording to claim 8 wherein the olefinic compound and the metal chelateare linked to separate molecules of the linking group to which thebinding partner for the analyte is non-covalently bound and wherein thelinking group may be the same or different.
 11. The chemiluminescentreagent according to claim 1 wherein the hydrophilic macromolecule is aprotein.
 12. The chemiluminescent reagent according to claim 7 whereinthe specific binding pair is selected from the group consisting of (i)small molecule and binding partner for the small molecule and (ii) largemolecule and binding partner for the large molecule.
 13. Thechemiluminescent reagent according to claim 1 wherein the metal of themetal chelate is a rare earth metal or a metal of Group VIII.
 14. Thechemiluminescent reagent according to claim 1 wherein the metal isselected from the group consisting of europium, terbium, dysprosium,samarium osmium and ruthenium.
 15. The chemiluminescent reagentaccording to claim 1 wherein the metal chelate comprises a chelatingagent selected from the group consisting of NHA, BHHT, BHHCT, DPP, TTA,NPPTA, NTA, TOPO, TPPO, BFTA, 2,2-dimethyl-4-perfluorobutyoyl-3-butanone(fod), 2,2′-dipyridyl (bpy), bipyridylcarboxylic acid, aza crown ethers,aza cryptands and trioctylphosphine oxide and derivatives thereof. 16.The chemiluminescent reagent according to claim 1 wherein the olefiniccompound is selected from the group consisting of thioxenes, dioxenes,enol ethers, enamines, 9-alkylidenexanthans,9-alkylidene-N-alkylacridans, aryl vinyl ethers, arylimidazoles andlucigenin and derivatives thereof.
 17. The chemiluminescent reagentaccording to claim 1 wherein the metal is europium, the chelating agentis BHHCT and the olefinic compound is a carboxyl derivative of thioxene.18.-21. (canceled)